diversity and phylogenetic relationships of hemosporidian parasites in ...

J. Parasitol., 99(2), 2013, pp. 270–276 Ó American Society of Parasitologists 2013

DIVERSITY AND PHYLOGENETIC RELATIONSHIPS OF HEMOSPORIDIAN PARASITES IN ´ BIRDS OF SOCORRO ISLAND, MEXICO, AND THEIR ROLE IN THE RE-INTRODUCTION OF THE SOCORRO DOVE (ZENAIDA GRAYSONI ) nas†, Claire Loiseau, Douglas A. Bell‡, and Ravinder N. M. Jenny S. Carlson, Juan E. Mart´ınez-Go´mez*, Gediminas Valkiu Sehgal Department of Biology, San Francisco State University, San Francisco, California 94132. Correspondence should be sent to: [email protected] ABSTRACT:

The Socorro dove Zenaida graysoni, endemic to Socorro Island, was last reported in the wild in 1972. Fortunately, the species has been propagated in zoos in Europe and the United States, and plans are under way to re-introduce it to its native habitat. This will be the first known attempt to return a bird species extinct in the wild to its ancestral island. In order to assess the disease threats the Socorro dove may face, the avifauna of Socorro Island, with a specific focus on Socorro ground doves Columbina passerina socorroensis and mourning doves Zenaida macroura, as well as Socorro doves in captivity, were screened for blood parasites of the genera Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma spp. We found Haemoproteus spp. in 17 (74%) of 23 Socorro ground doves, 23 (92%) of 25 mourning doves, and 3 (14%) of 21 northern mockingbirds; none of the other bird species showed infections. Here, we report the phylogenetic analysis of 19 distinct lineages of Haemoproteus spp. detected in birds of Socorro Island and compare their evolutionary relationships to parasites detected in the avifauna of the Gala´pagos Islands, continental Latin America, and Europe. Microscopic examination revealed 1 mourning dove infected with Plasmodium (Haemamoeba), thus underscoring the importance of using both PCR and microscopy when analyzing avian blood samples for hemosporidian parasites. The study confirms that the Socorro dove will most likely be exposed to Haemoproteus spp. that currently infect mourning doves and Socorro ground doves of Socorro Island. A monitoring program for both birds and vectors should be implemented to establish the prevalence of Plasmodium sp. and as a necessary conservation measure for critically endangered birds on the island.

(Nyctanassa violacea gravirostris) (Rodriguez-Estrella et al., 1996). Two avian species arrived on the island recently, the mourning dove (Zenaida macroura) and the northern mockingbird (Mimus polyglottos) (Jehl and Parkes, 1982), while 2 endemic taxa are no longer on the island, the Socorro dove (Zenaida graysoni) and the elf owl (Micrathene whitneyi graysoni) (Jehl and Parkes, 1982; Rodr ´ıguez-Estrella et al., 1996). The extinction of the endemic elf owl is definitive, but the Socorro dove currently survives in captivity (Mart ´ınez-Go´mez et al., 2010). Cats and humans contributed to the decline of the endemic dove, last reported on the island in 1972 (VelascoMurgia, 1982; Jehl and Parkes, 1983). Ongoing collaborative efforts are in place to achieve its re-introduction. At this point in time, several threats remain that could hinder these efforts, including the presence of potential predators (introduced cats and native hawks), the presence of the mourning doves (which may hybridize with the Socorro dove), and pathogens currently found on the island. Although there is no evidence that blood parasites contributed to the extinction of the Socorro dove, they could potentially impede their successful re-introduction (e.g., Marzal et al., 2005; Donovan et al., 2008; Puente et al., 2010; Olias et al., 2011). This is especially true when considering that animals that have been bred in captivity may not be immunologically competent to resist an infection caused by parasites or viruses (Viggers et al., 1993). Hemosporidians, including species of Plasmodium, Haemoproteus, and Leucocytozoon, are related vector-borne blood parasites typically found in reptiles, birds, and mammals (Valki unas, 2005). Plasmodium species are pathogenic and often cause disease in wild birds (Warner, 1968; Van Riper et al., 1986; Atkinson, 1999; Kilpatrick et al., 2006; Atkinson and LaPointe, 2009; Knowles et al., 2009, 2010). Haemoproteus species, which in some cases appear to be less pathogenic than Plasmodium, may still reduce host fitness (Allander, 1997; Knowles et al., 2009; Puente et al., 2010), cause severe diseases in avian hosts (Miltgen et al., 1981; Atkinson et al., 1988; Cardona et al., 2002), and even lead to death in wild birds (Valki unas, 2005; Donovan et al., 2008).

Socorro Island is the largest of the 4 Revillagigedo Islands, located approximately 700 km west of the port city of Manzanillo, Colima, Me´xico. Because of its isolation from the mainland, there is a high degree of biotic endemism (Brattstrom, 1990). Vertebrate wildlife diversity is low, with terrestrial birds predominating among a few reptiles and very few non-endemic mammals (Brattstrom, 1990). A major concern for vertebrate populations on Socorro Island, as for many other island populations, is the introduction of pathogens to immunologically naive species. For example, the introduction of avian malaria (Plasmodium relictum) contributed to major declines and extinctions in the avifauna of Hawaii (Warner, 1968; Van Riper et al., 1986; Van Riper et al., 2002), and may negatively affect the avifauna of the Gala´pagos Islands in the future (Padilla et al., 2004; Parker et al., 2006; Levin et al., 2009; Santiago-Alarcoń et al., 2012). Disease monitoring is essential for the conservation of island populations in order to prevent the spread of introduced pathogens that may cause the extirpation of one or more species. Such efforts are extremely important on Socorro Island because of the concentration of critically endangered bird species (Mart ´ınez-Go´mez and Curry, 1996; Mart ´ınez-Go´mez et al., 2001; Mart ´ınez-Go´mez and Jacobsen, 2004; Mart ´ınez-Go´mez et al., 2010). Socorro Island’s terrestrial avifauna includes 8 endemic species, i.e., the green parakeet (Aratinga holochlora brevipes), Socorro red-tailed hawk (Buteo jamaicensis socorroensis), Socorro ground dove (Columbina passerina socorroensis), Socorro wren (Troglodytes sissonii), Socorro mockingbird (Mimus graysoni), Socorro towhee (Pipilo maculatus socorroensis), tropical parula (Parula pitiayumi graysoni), and the yellow-crowned night heron Received 17 May 2012; revised 1 October 2012; accepted 8 October 2012. * Instituto de Ecolog ´ıa, A. C., Red de Interacciones Multitro´ficas, Apartado Postal 63, Xalapa, Veracruz 91000, Me´xico. † Nature Research Centre, Akademijos 2, Vilnius LT-08412, Lithuania. ‡ Dept. Ornithology and Mammalogy, California Academy of Sciences, 55 Music Concourse Drive, Golden Gate Park, San Francisco, California 95819. DOI: 10.1645/GE-3206.1 270

CARLSON ET AL.—DIVERSITY AND PHYLOGENETIC RELATIONSHIPS OF MEXICAN HEMOSPORIDEANS

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(Kerlin, 1964) and immediately stored in lysis buffer (10 mM Tris-HCL, pH 8.0, 100 mM EDTA, 2% SDS) at room temperature while on the island and, upon return to the lab at San Francisco State University, were then stored at 20 C. Additionally, we acquired 12 blood samples from Socorro doves held captive at the Albuquerque Zoo, New Mexico. Parasite screening using microscopy Two blood films were prepared from each bird. Blood films were airdried within 5–10 sec after their preparation; they were fixed in absolute methanol in the field and then stained with Giemsa in the laboratory as described by Valki unas (2005). We examined nearly all blood films using microscopy. Some of the blood smears were of insufficient quality to yield reliable results. An Olympus BX61 light microscope equipped with Olympus DP70 digital camera and imaging software Analysis Five was used to examine slides. One blood film from each bird was examined for 10–15 min at low magnification (3400), and then at least 100 fields were studied at high magnification (31,000). Voucher blood films were deposited in the Nature Research Centre, Vilnius, Lithuania. Parasite screening using PCR

FIGURE 1. Map of Socorro Island showing the geographical distribution of study sites 1–4 where birds were trapped (Base map courtesy of Manuel Escamilla—INECOL, according to the photomap prepared by Me´xico’s National Institute of Statistics, Geography and Informatics— INEGI).

A preliminary survey was conducted on Socorro Island for avian pathogens in the 2 columbid species currently present on the island (Yanga et al., 2011), i.e., the endemic Socorro ground dove, which is found in small numbers at lower elevations throughout the island (Wehtje et al., 1993), and the mourning dove, a species that arrived to the island in the 1970s (Jehl and Parkes, 1982). The mourning doves are congeneric with the Socorro dove (Johnson and Clayton, 2000) and colonized the island as the Socorro dove population was waning (Jehl and Parkes, 1983); the Socorro ground dove coexisted with the Socorro dove (Jehl and Parkes, 1982). The objectives of the present study were as follows: (1) to screen the avifauna of Socorro Island for parasites, including species of Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma; (2) to perform a phylogenetic analysis of the parasites of Socorro Island using a portion of the mtDNA cyt b gene to establish lineage relationships with parasites from other regions of the world; and (3) to screen Socorro doves residing at the Albuquerque Zoo in New Mexico, which will be used in the reintroduction program. MATERIALS AND METHODS

Parasite DNA was extracted from avian whole blood stored in lysis buffer, including the 12 Socorro dove samples from the zoo, following animal tissue protocols of the Wizard SV Genomic DNA Purification kits (Promega Corporation, Madison, Wisconsin). All polymerase chain reactions (PCR) were carried out in a 25 ll reaction mixture containing 10–100 ng of genomic DNA (2 ll of template DNA), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3.0 mM MgCl, 0.4 mM of each dNTP, 0.4 mM of each primer, 5 ll of CL buffer, and 0.5 units Taq (Qiagen, Valencia, California). The extracted avian blood samples were then screened for species of Trypanosoma, Leucocytozoon, Plasmodium, and Haemoproteus. Screening of Trypanosoma spp. was conducted by a nested PCR with primers Tryp763/Tryp1016 and Tryp99/Tryp957 designed and used by Valki unas et al. (2011). Screening of Leucocytozoon spp. was conducted by a nested PCR with primers described in Hellgren et al. (2004) HaemNR3/ HaemNFI and HaemFL/HaemR2L. To screen for Plasmodium and Haemoproteus spp., we amplified ~740 bp of the mitochondrial cytochrome oxidase subunit b gene (cyt b) using 2 sets of primers. The first set were L15183 and the H15730 developed by Fallon et al. (2003) and Szymanski and Lovette (2005) as employed by Chasar et al. (2009). The second primer set consisted of the nested primers HaemNF/HaemNR2 and HaemF/HaemR2 as described in Waldenstrom ¨ et al. (2004). All PCR products were viewed on 1.8% agarose gels stained with ethidium bromide. Positive PCR products were then sent to Elim Biopharmaceuticals Inc., Hayward, California, for Bi-directional sequencing and were edited using Sequencher 4.8 (GeneCodes, Ann Arbor, Michigan). Parasite sequences that differed by only 1 to 3 base pairs were considered a separate distinct lineage (Ricklefs and Fallon, 2002). Distinct lineages were then verified by repeating an independent PCR and sequencing analysis. Additionally, chromatograms of all sequences were inspected for double peaks to ensure that no individual harbored multiple infections. If multiple peaks were found in the same chromatograms of 1 blood sample, it was considered to be infected with multiple distinct lineages (Pe´rez-Tris and Bensch, 2005). In addition to the samples collected in 2009, we obtained 12 DNA positive samples from the study conducted in 2004 by Yanga et al. (2011) from 6 mourning doves and 6 Socorro ground doves; the cyt b gene was amplified using the nested PCR protocol from Waldenstrom ¨ et al. (2004). All final sequences were deposited to GenBank with accession numbers (JN788932–JN788950).

Sample collection

Phylogenetic analysis

Sampling took place during 3–19 July 2009 at 4 sites on the island (Fig. 1): Site 1 Las Grutas (18844 0 4.8 00 N, 110856 0 48.3 00 W); Site 2 Los Cedros (18845 0 28.9 00 N, 110856 0 44.4 00 W); Site 3 Playa Blanca (18848 0 51 00 N, 111802 0 422.5 00 W); and Site 4 Llano de Borregos (18845 0 57.5 00 N, 110856 0 58.5 00 W). Birds were trapped using 10 to 12 mist nets (12 m long and 3 m tall, with 36 3 36 mm and 60 3 60 mm mesh), which were set up in the morning shortly after sunrise and checked every 20 min until dusk. Upon capture, each bird was identified, measured, and weighed. Additionally, numbered aluminum bands were placed on each bird. Blood samples of 5 to 20 ll were collected by brachial venipuncture

Phylogenetic relationships were analyzed using the best fit GTRþG model of molecular evolution as calculated with MrModeltest (Nylander, 2004), incorporating results in MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001). In addition to Haemoproteus sequences obtained from Socorro Island, 37 Haemoproteus sequences from the Gala´pagos Islands, North America, and continental Latin America (Valki unas et al., 2010) were obtained from Genbank (see Fig. 2 for accession numbers). Two Leucocytozoon schoutedeni lineages were used as outgroups. Two Markov Chain Monte Carlo (MCMC) simulations were run simultaneously for 10 million generations with sampling every 200 generations, creating 100,000

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FIGURE 2. Bayesian phylogeny of 45 mitochondrial cytochrome b Haemoproteus spp. lineages found in birds from Socorro Island, the Gala´pagos Islands, and continental Latin America. Two Leucocytozoon schoutedeni lineages were used as outgroups. Gray boxes indicate a grouping of closely related lineages of hemoproteids. The Bayesian posterior probabilities are depicted at each node. Lineages obtained from Socorro Island are in bold. All other Haemoproteus lineages are delineated by the parasite name followed by the Genbank accession numbers. Clade A, species of the subgenus Parahaemoproteus; clades B and C, species of the subgenus Haemoproteus.


TABLE I. Sample numbers of Haemoproteus spp. lineages found in birds on Socorro Island in 2004 and 2009 (N ¼ overall number of recorded infections, n ¼ numbers of recorded infections in each species). Bird species (n)

Lineages

N

Mourning doves

Socorro ground doves

Mockingbirds

SocH1 SocH2 SocH3 SocH4 SocH5 SocH6 SocH7 SocH8 SocH9 SocH10 SocH11 SocH12 SocH13 SocH14 SocH15 SocH16 SocH17 SocH18 SocH19 Total

13 8 11 8 2 1 1 2 1 1 1 2 2 1 1 2 1 2 2 62

13 3 0 0 2 1 0 0 1 1 1 2 2 1 1 2 1 2 2 35

0 3 11 8 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 24

0 2 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 3

trees. The first 25,000 trees were discarded from the sample as the ‘‘burnin’’ period that accounted for 25% of the trees. The remaining trees were used to construct a majority rule consensus tree and to calculate the posterior probabilities of the individual clades (Labarthe et al., 1998). The genetic distances between different lineages were calculated in PAUP Version 4.0 (Swofford, 2001).

RESULTS Parasite diversity and prevalence In total, 122 birds were caught and sampled for processing by PCR. Included were 21 northern mockingbirds, 7 Socorro wrens, 1 Socorro mockingbird, 40 tropical parulas, 5 Socorro towhees, 25 mourning doves, and 23 Socorro ground doves. We screened all birds for blood parasites and found 46 individuals infected, i.e., 23 (92%) mourning doves, 17 (74%) Socorro ground doves, and 3 (14%) northern mockingbirds. All other species tested negative for parasite DNA. Of the 12 lineages from the 2004 study, 8 aligned with lineages that were detected in the present study (SocH1, SocH2, SocH3, SocH4, SocH5, SocH8, SocH13, and SocH16), while 4 lineages were distinct (SocH9, SocH10, SocH15, and SocH17). We detected 19 distinct lineages of Haemoproteus spp. (Table I) in the 58 combined sequences from the 2004 and the 2009 studies. No species of Leucocytozoon, Plasmodium, or Trypanosoma were detected in any bird species by PCR. Microscopic examination confirmed these results, with the following exceptions. Two of the mourning doves, which were negative by microscopy, tested positive by PCR and aligned with the SocH1 lineage. PCR examination revealed that only 6 mourning doves were co-infected with different species of Haemoproteus, while microscopic examination revealed that 18 of 21 mourning doves (85%) were co-infected with Haemoproteus columbae and Haemoproteus multipigmentatus, which included the

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6 that were detected by PCR. Blood samples that were deemed to be co-infected by PCR were re-extracted, and PCR and sequencing were conducted as described above. We successfully obtained a clean sequence for 1 of the parasites in the co-infected bird after re-extraction. Most interesting, however, was the finding by microscopy that, in addition to being co-infected with both H. columbae and H. multipigmentatus, 1 mourning dove also harbored Plasmodium (Haemamoeba) spp. The 12 Socorro dove blood samples tested negative for all parasites by PCR. Blood slides were not available for these individuals. Phylogenetic relationships Phylogenetic relationships among the 19 Haemoproteus lineages found in birds of Socorro Island and those found in the Gala´pagos Islands and in the Americas are depicted in Figure 2. Three distinct clades were observed, i.e., Group A, Group B, and Group C. Group A consisted of 21 closely related lineages belonging to the subgenus Parahaemoproteus and 1 lineage from Socorro Island that was obtained from a northern mockingbird (SocH7). Group B consisted of 15 Haemoproteus (Haemoproteus) spp. lineages obtained from mourning doves of Socorro Island, 3 H. columbae lineages, and 13 lineages of H. multipigmentatus obtained from doves captured in the Gala´pagos, Me´xico, Guatemala, and Peru. ´ Genetic distances within the highly supported clade containing H. multipigmentatus and the Haemoproteus lineages from Socorro Island ranged from 0.6 to 4.8%. Upon microscopic examination of blood smears, it was determined that all Socorro Island lineages in Group B belong to morphospecies H. multipigmentatus (Valki unas et al., 2010). It is interesting to note that lineage SocH2 was the only one found in multiple bird species, i.e., 3 mourning doves, 3 Socorro ground doves, and 2 northern mockingbirds. Finally, Group C consisted of a monophyletic group of 3 Haemoproteus lineages that were detected in Socorro ground doves. Based on morphological evidence, parasites of these lineages belong to a previously undescribed species, which will be described elsewhere. Genetic distances between lineages found in Group B and Group C ranged from 7.1 to 9.8% (Valki unas et al., 2013). DISCUSSION Haemoproteus spp. were identified both by PCR and microscopy from blood samples of 3 avian species, i.e., the Socorro ground dove, the mourning dove, and the northern mockingbird. Our study provides the first documentation of the phylogenetic relationships of hemosporidian parasites in the avifauna populations of Socorro Island. We found 19 lineages of Haemoproteus spp., of which the majority were identified via microscopic and sequence analysis as H. multipigmentatus. This indicates that the diversity of these parasites on Socorro Island is large and includes at least 1 undescribed Haemoproteus parasite (lineages of the clade C). Group A in Figure 2 depicts an alignment of closely related lineages belonging to the subgenus Parahaemoproteus. This grouping also includes a distinct lineage (SocH7) that was detected in a northern mockingbird. Lineage SocH7 fell in a highly supported clade with Haemoproteus cyanomitrae, a species described by Iezhova et al. (2010), which was detected in an African olive sunbird. We did not manage to identify this parasite

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to species level because of light parasitemia (a few immature gametocytes were seen). In order to understand where this parasite may have originated, additional sampling and identification of morphospecies must be conducted on hemoproteids on the mainland Americas. Group B (Fig. 2) depicts a grouping of 3 lineages of H. columbae with lineages of H. multipigmentatus and Haemoproteus sp. detected primarily in mourning doves of Socorro Island. The high support of the clade in Group B containing H. multipigmentatus lineages found in doves from the Gala´pagos Islands, continental Latin America, and the Socorro Island Haemoproteus lineages suggests that these parasites may be the same species. Microscopic examination of the slides revealed gametocytes that are indistinguishable from H. multipigmentatus. The records of this parasite in Socorro Island represent a new avian host for this species. There is no geographical differentiation between parasites from Socorro Island and those from continental populations and the Gala´pagos Islands. Santiago-Alarcoń et al. (2010) observed a similar pattern in their phylogenetic analyses. Because Socorro Island H. multipigmentatus did not form a unique clade, but is intermixed with lineages from the mainland and the Gala´pagos Island, we can speculate that this morphospecies has a broad distribution and must have been brought recently by mourning doves, northern mockingbirds, or both. Group C (Fig. 2) depicts a monophyletic group of 3 distinct lineages that were obtained solely from Socorro ground doves. Morphologically, all 3 lineages of clade C represent the same morphospecies, which is different from H. columbae and H. multipigmentatus and will be described elsewhere (G. Valki unas, unpubl. obs.). The placement of this clade in this phylogenetic tree suggests that Socorro ground doves harbor different Haemoproteus species than those detected in mourning doves. However, there is 1 exception, SocH2, which was a Haemoproteus sp. detected in the Socorro ground dove, the mourning dove, and the northern mockingbird. This suggests that this species of Haemoproteus may be a generalist, capable of infecting multiple bird species, as is frequently the case in some avian hemoproteids (Beadell et al., 2004, 2009; Krizanauskien_ ˇ e et al., 2010; Loiseau et al., 2010). The present study, in addition to providing an account of the diversity of Haemoproteus spp. on Socorro Island, has provided further evidence of the importance of combining both PCR and microscopy methods when analyzing bird blood samples for hemosporidian parasites (Valki unas et al., 2008). No Plasmodium, Leucocytozoon, or Trypanosoma spp. were detected in any of the birds by PCR-based methods. Plasmodium (Haemamoeba) sp. infection was detected by microscopy in a single mourning dove. However, microscopic examination of the blood smears revealed multiple co-infections and 1 infection by Plasmodium sp. The primers used in this study appear to preferentially bind to cyt b gene sequences of H. multipigmentatus when in the presence of a co-infection of multiple Haemoproteus spp. This indicates that when using PCR as a diagnostic method to determine hemosporidian diversity in avian populations, multiple genes of the parasite should be analyzed, and primers that are selective for 1 species over another should be used with caution (Valki unas et al., 2010). New methods to address co-infections within bird populations should be considered and explored while still continuing to use both PCR and microscopy methods concurrently to ensure that each hemosporidian species is accounted for.

This study was carried out in conjunction with a study of the mosquito fauna of Socorro Island (Carlson et al., 2011) to determine the diversity of mosquito species and whether or not they harbored hemosporidian parasites. Haemoproteus spp. lineages have been detected in wild caught mosquitoes (Ishtiaq et al., 2008; Njabo et al., 2009). However there is no current evidence that mosquitoes can transmit Haemoproteus spp. Carlson et al. (2011) found no mosquitoes infected with Haemoproteus but did find mosquitoes infected with Plasmodium spp. This study underscores the need to monitor the insect vectors as well as the bird hosts; had we relied solely on PCR-based detection of Plasmodium spp. in birds, we would have concluded erroneously that Plasmodium was not present on the island. In sum, this phylogenetic analysis of Haemoproteus spp. provides a more extensive understanding of the diversity of these parasites in bird populations of Socorro Island and some insight into what the Socorro doves may face upon re-introduction. Based upon the results of this study, as suggested by Yanga et al. (2011), we expect that the Socorro doves will most likely be exposed to the Haemoproteus spp. that currently infect the mourning doves and the Socorro ground doves of Socorro Island. This is a plausible scenario when considering earlier documentation of the low host specificity of several lineages of Haemoproteus parasites (Krizanauskien_ ˇ e et al., 2010) and that lineage SocH2 was recovered from blood samples of mourning doves, Socorro ground doves, and northern mockingbirds. Previous studies have shown that H. columbae, a common parasite of birds of the order Columbiformes, can be lethal to doves (Earle´ et al., 1993) and that Haemoproteus infections can also affect the health and fitness of passerine birds (Merino et al., 2000; Marzal et al., 2005). In addition, mortality due to Haemoproteus spp. infections has been reported in birds in American zoos (Ferrell et al., 2007) and captive birds in Europe (Olias et al., 2011). Thus, hemoproteid infections warrant more attention in conservation projects, especially with potentially naive bird populations. A continuous monitoring program of both the avian and insect vector populations using PCR-based and microscopy techniques on Socorro Island is required to understand how parasites interact with both the host and potential vectors. In conjunction with ongoing programs for population monitoring, this approach would provide a complete picture of all possible threats that could jeopardize the success of the re-introduction program for the Socorro dove and the population viability of other already fragile endemic avifauna. ACKNOWLEDGMENTS We would like to offer our sincere gratitude and recognition to the Secretar ´ıa de Marina–Armada de Me´xico, and, in particular, to Admiral Ezequiel Ramiro Bordonave, Captain Pedro Villaciz Leoń, and the military personnel stationed at Socorro Island for their hospitality and valuable logistical support. Tatjana A. Iezhova is gratefully acknowledged for help with microscopic examination of blood films. Patricia Escalante at IBUNAM kindly provided access to samples collected in 2004. We would like to thank the members of the Island Endemics Foundation for their support and technical advice. We would also like to extend our gratitude to the Albuquerque Zoo in New Mexico for providing blood samples from captive Socorro doves. Funding was provided by the College of Science and Engineering, San Francisco State University, the American Society of Parasitologists Willis A. Reid Student Research Grant, the San Francisco State University Instructionally Related Activities Grant, Conservation International, the American Bird Conservancy, the Islands Endemics Foundation, and INECOL. We would also like to thank Dr. Susan


Perkins and 2 anonymous reviewers whose comments and suggestions improved this manuscript. All research was carried out under permits (SGPA/DGVS/51015/03 and SGPA/DGVS/04005/08) issued by the Secretar ´ıa de Medio Ambiente y Recursos Naturales and the Secretar ´ıa de Gobernacioń. Research protocols in this study also comply with IACUC permit A9-002 SFSU.

LITERATURE CITED ALLANDER, K. 1997. Reproductive investment and parasite susceptibility in the great tit. Functional Ecology 11: 358–364. ATKINSON, C. T. 1999. Hemosporidiosis. Field manual of wildlife diseases: General field procedures and diseases of birds. Biological Resources Division Information and Technology Report 1: 193–199. ———, D. J. FORRESTER, AND E. C. GREINER. 1988. Pathogenicity of Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in experimentally infected domestic turkeys. Journal of Parasitology 74: 228–239. ———, AND D. A. LAPOINTE. 2009. Introduced avian diseases, climate change, and the future of Hawaiian honeycreepers. Journal of Avian Medicine and Surgery 23: 53–63. BEADELL, J. S., R. COVAS, C. GEBHARD, F. ISHTIAQ, M. MELO, B. K. SCHMIDT, S. L. PERKINS, G. R. GRAVES, AND R. C. FLEISCHER. 2009. Host associations and evolutionary relationships of avian blood parasites from West Africa. International Journal for Parasitology 39: 257–266. ———, E. GERING, J. AUSTIN, J. P. DUMBACHER, M. A. PEIRCE, T. K. PRATT, C. T. ATKINSON, AND R. C. FLEISCHER. 2004. Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region. Molecular Ecology 13: 3829–3844. BRATTSTROM, B. H. 1990. Biogeography of the Islas Revillagigedo, Me´xico. Journal of Biogeography 17: 177–183. CARDONA, C. J., A. IHEJIRIKA, AND L. MCCLELLAN. 2002. Haemoproteus lophortyx infection in bobwhite quail. Avian Diseases 46: 249–255. ´ -GO´MEZ, A. CORNEL, C. LOISEAU, AND R. N. CARLSON, J. S., J. E. MARTINEZ M. SEHGAL. 2011. Implications of Plasmodium parasite infected mosquitoes on an insular avifauna: The case of Socorro Island, Me´xico. Journal of Vector Ecology 36: 213–219. , T. IEZHOVA, T. B. SMITH, AND R. CHASAR, A., C. LOISEAU, G. VALKIUNAS N. M. SEHGAL. 2009. Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats. Molecular Ecology 18: 4121–4133. DONOVAN, T. A., M. SCHRENZEL, T. A. TUCKER, A. P. PESSIER, AND I. H. STALIS. 2008. Hepatic hemorrhage, hemocoelom, and sudden death due to Haemoproteus infection in passerine birds: Eleven cases. Journal of Veterinary Diagnostic Investigation 20: 304–313. EARLE´, R. A., S. S. BATIANELLO, G. F. BENNET, AND R. C. KRECEK. 1993. Histopathology and morphology of the tissue stages of Haemoproteus columbae causing mortality in Columbiformes. Avian Pathology 22: 67–80. FALLON, S. M., E. BERMINGHAM, AND R. E. RICKLEFS. 2003. Island and taxon effects in parasitism revisited: Avian malaria in the Lesser Antilles. Evolution 57: 606–615. FERRELL, S. T., K. SNOWDEN, A. B. MARLAR, M. GARNER, AND N. P. LUNG. 2007. Fatal hemoprotozoal infections in multiple avian species in a zoological park. Journal of Zoo and Wildlife Medicine 38: 309–316. ¨ , AND S. BENSCH. 2004. A new PCR assay HELLGREN, O., J. WALDENSTROM for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. Journal of Parasitology 90: 797–802. HUELSENBECK, J. P., AND F. RONQUIST. 2001. MrBayes: A program for the Bayesian inference of phylogeny. Bioinformatics 17: 754–755. , C. LOISEAU, T. B. SMITH, AND R. N. M. IEZHOVA, T. A., G. VALKIUNAS SEHGAL. 2010. Haemoproteus cyanomitrae sp. nov. (Haemosporida: Haemoproteidae) from a widespread African songbird, the olive sunbird Cyanomitra olivacea. Journal of Parasitology 96: 137–143. ISHTIAQ, F., L. GUILLAUMOT, S. M. CLEGG, A. B. PHILLIMORE, R. A. BLACK, I. P. F. OWENS, N. I. MUNDY, AND B. C. SHELDON. 2008. Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands. Molecular Ecology 17: 4545–4555. JEHL, J. R., AND K. C. PARKES. 1982. The status of the avifauna of the Revillagigedo Islands, Mexico. The Wilson Bulletin 94: 1–19. ———, AND ———. 1983. ‘‘Replacements’’ of landbird species on Socorro Island, Mexico. The Auk 100: 551–559.

275

JOHNSON, K. P., AND D. H. CLAYTON. 2000. Nuclear and mitochondrial genes contain similar phylogenetic signal for pigeons and doves (Aves: Columbiformes). Molecular Phylogenetics and Evolution 14: 141–151. KERLIN, R. E. 1964. Venipuncture of small birds. Journal of the American Veterinary Medical Association 144: 870–874. KILPATRICK, A. M., D. A. LAPOINTE, C. T. ATKINSON, B. L. WOODWORTH, J. K. LEASE, M. E. REITER, AND K. GROSS. 2006. Effects of chronic avian malaria (Plasmodium relictum) infection on reproductive success of Hawaii Amakihi (Hemignathus virens). The Auk 123: 764–774. KNOWLES, S. C. L., S. NAKAGAWA, AND B. C. SHELDON. 2009. Elevated reproductive effort increases blood parasitaemia and decreases immune function in birds: A meta regression approach. Functional Ecology 23: 405–415. ———, V. PALINAUSKAS, AND B. C. SHELDON. 2010. Chronic malaria infections increase family inequalities and reduce parental fitness: Experimental evidence from a wild bird population. Journal of Evolutionary Biology 23: 557–569. ˇ E_ , A., J. PE´REZ-TRIS, V. PALINAUSKAS, O. HELLGREN, S. KRIZANAUSKIEN . 2010. Molecular phylogenetic and BENSCH, AND G. VALKIUNAS morphological analysis of haemosporidian parasites (Haemosporida) in a naturally infected European songbird, the blackcap Sylvia atricapilla, with description of Haemoproteus pallidulus sp. nov. Parasitology 137: 217–227. LABARTHE, N., M. L. SERRAÕ, Y. F. MELO, S. J. OLIVEIRA, AND R. ¸ -DE-OLIVEIRA. 1998. Potential vectors of Dirofilaria immitis LOURENCO (Leidy, 1856) in Itacoatiara, oceanic region of Niteroí municipality, State of Rio de Janeiro, Brazil. Memo´rias do Instituto Oswaldo Cruz 93: 425–432. LEVIN, I. I., D. C. OUTLAW, F. H. VARGAS, AND P. G. PARKER. 2009. Plasmodium blood parasite found in endangered Galapagos penguins (Spheniscus mendiculus). Biological Conservation 142: 3191–3195. , A. CHASAR, A. HUTCHINSON, W. LOISEAU, C., T. IEZHOVA, G. VALKIUNAS BUERMANN, T. B. SMITH, AND R. N. M. SEHGAL. 2010. Spatial variation of haemosporidian parasite infection in African rainforest bird species. Journal of Parasitology 96: 21–29. ´ -GO´MEZ, J. E., AND R. L. CURRY. 1996. The conservation status MARTINEZ of the Socorro mockingbird Mimodes graysoni in 1993–1994. Bird Conservation International 6: 271–283. ———, A. FLORES-PALACIOS, AND R. L. CURRY. 2001. Habitat requirements of the Socorro mockingbird Mimodes graysoni. Ibis 143: 456– 467. ———, H. M. HORBLIT, S. G. STADLER, AND P. W. SHANNON. 2010. Reintroduction of the Socorro dove, Socorro Island, Revillagigedo Archipelago, Mexico. In Global re-introduction perspectives, Volume II, P. S. Soorae (ed.). The IUCN/SSC Re-introduction Specialist Group (RSG), Abu Dhabi, UAE, p. 182–186. ———, AND J. K. JACOBSEN. 2004. The conservation status of Townsend’s shearwater Puffinus auricularis auricularis. Biological Conservation 116: 35–47. MARZAL, A., F. DE LOPE, C. NAVARRO, AND A. P. MLLER. 2005. Malarial parasites decrease reproductive success: An experimental study in a passerine bird. Oecologia 142: 541–545. MERINO, S., J. MORENO, J. J. SANZ, AND E. ARRIERO. 2000. Are avian blood parasites pathogenic in the wild? A medical experiment in blue tits (Parus caeruleus). Proceedings of the Royal Society of London, Series B 267: 2507–2510. MILTGEN, F., I. LANDAU, N. RATANAWORABHAN, AND S. YENBUTRA. 1981. Parahaemoproteus desseri n. sp. Game´togonie et schizogonie chez l’hote ˆ naturel: Psittacula roseata de Thailande, et sporogonie expe´rimentale chez Culicoides nubeculosus. Annales de Parasitologie Humaine et Compareé 56: 123–130. NJABO, K. Y., A. J. CORNEL, R. N. M. SEHGAL, C. LOISEAU, W. BUERMANN, , AND T. B. SMITH. 2009. R. J. HARRIGAN, J. POLLINGER, G. VALKIUNAS Coquillettidia (Culicidae, Diptera) mosquitoes are natural vectors of avian malaria in Africa. Malaria Journal 8: 193. NYLANDER, J. A. A. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University 2, Uppsala, Sweden. OLIAS, P., M. WEGELIN, W. ZENKER, S. FRETER, A. D. GRUBER, AND R. KLOPFLEISCH. 2011. Avian malaria deaths in parrots, Europe. Emerging Infectious Diseases 17: 950–952.

276


PADILLA, L. R., D. SANTIAGO-ALARCOŃ, J. MERKEL, R. E. MILLER, AND P. G. PARKER. 2004. Survey for Haemoproteus spp., Trichomonas gallinae, Chlamydophila psittaci, and Salmonella spp. in Galapagos Islands Columbiformes. Journal of Zoo and Wildlife Medicine 35: 60–64. PARKER, P. G., N. K. WHITEMAN, AND R. E. MILLER. 2006. Conservation medicine on the Gala´pagos Islands: Partnerships among behavioral, population, and veterinary scientists. The Auk 123: 625–638. PE´REZ-TRIS, J., AND S. BENSCH. 2005. Dispersal increases local transmission of avian malaria parasites. Ecology Letters 8: 838–845. PUENTE, J. M., S. MERINO, G. TOMA´S, J. MORENO, J. MORALES, E. LOBATO, ´ -FRAILE, AND E. J. BELDA. 2010. The blood parasite S. GARCIA Haemoproteus reduces survival in a wild bird: A medication experiment. Biology Letters 6: 663–665. RICKLEFS, R. E., AND S. M. FALLON. 2002. Diversification and host switching in avian malaria parasites. Proceedings of the Royal Society of London Series B: Biological Sciences 269: 885–892. RODRIGUEZ-ESTRELLA, R., J. L. LEON DE LA LUZ, A. BRECEDA, A. CASTELLANOS, J. CANCINO, AND J. LLINAS. 1996. Status, density and habitat relationships of the endemic terrestrial birds of Socorro Island, Revillagigedo Islands, Mexico. Biological Conservation 76: 195–202. SANTIAGO-ALARCOŃ, D., D. C. OUTLAW, R. E. RICKLEFS, AND P. G. PARKER. 2010. Phylogenetic relationships of haemosporidian parasites in New World Columbiformes, with emphasis on the endemic Galapagos dove. International Journal for Parasitology 40: 463–470. ———, R. E. RICKLEFS, AND P. G. PARKER. 2012. Parasitism effects on the body condition of the endemic Gala´pagos Dove (Zenaida galapagoensis) and its relation to host genetic diversity and white blood cell counts. Studies in Avian Biology 42: 31–41. SWOFFORD, D. L. 2001. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.0b. Sinauer Associates, Sunderland, Massachusetts. SZYMANSKI, M. M., AND I. J. LOVETTE. 2005. High lineage diversity and host sharing of malarial parasites in a local avian assemblage. Journal of Parasitology 91: 768–774. , G. 2005. Avian malaria parasites and other haemosporidia. VALKIUNAS CRC Press, Boca Raton, Florida, 946 p. ———, T. A. IEZHOVA, J. S. CARLSON, AND R. N. M. SEHGAL. 2011. Two new Trypanosoma species from African birds, with notes on

taxonomy of avian trypanosomes. Journal of Parasitology 97: 924– 930. ´ ———, ———, E. EVANS, J. S. CARLSON, J. E. MARTINEZ -GO´MEZ, AND R. N. M. SEHGAL. 2013. Two new Haemoproteus species (Haemosporida: Haemoproteidae) from Columbiform birds. Journal of Parasitology 99. (In press). ˇ E_ , V. PALINAUSKAS, R. N. M. SEHGAL, ———, ———, A. KRIZANAUSKIEN AND S. BENSCH. 2008. A comparative analysis of microscopy and PCR-based detection methods for blood parasites. Journal of Parasitology 94: 1395–1401. ———, D. SANTIAGO-ALARCOŃ, I. LEVIN, T. IEZHOVA, G. PATRICIA, AND P. PARKER. 2010. A new Haemoproteus species (Haemosporida: Haemoproteidae) from the endemic Galapagos dove Zenaida galapagoensis, with remarks on the parasite distribution, vectors, and molecular diagnostics. Journal of Parasitology 96: 783–792. VAN RIPER, C., S. G. VAN RIPER, M. L. GOFF, AND M. LAIRD. 1986. The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecological Monographs 56: 327–344. ———, ———, AND W. R. HANSEN. 2002. Epizootiology and effect of avian pox on Hawaiian forest birds. The Auk 119: 929–942. VELASCO-MURGIA, M. 1982. Colima y las Isias de Revillagigedo. Universidad de Colima, Colima, Mexico, 150 p. VIGGERS, K. L., D. B. LINDENMAYER, AND D. M. SPRATT. 1993. The importance of disease in reintroduction programmes. Wildlife Research 20: 687–698. ¨ , J., S. BENSCH, D. HASSELQUIST, AND O. OSTMAN. 2004. A WALDENSTROM new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. Journal of Parasitology 90: 191–194. WARNER, R. E. 1968. The role of introduced diseases in the extinction of the endemic Hawaiian avifauna. Condor 70: 101–120. WEHTJE, W., H. S. WALTER, R. RODRIGUEZ-ESTRELLA, J. LLINAS, AND A. CASTELLANOS-VERA. 1993. An annotated checklist of the birds of Isla Socorro, Mexico. Western Birds 24: 1–16. ´ YANGA, S., J. E. MARTINEZ -GO´MEZ, R. N. M. SEHGAL, P. ESCALANTE, F. C. CAMACHO, AND D. A. BELL. 2011. A preliminary survey for avian pathogens in Columbiform birds on Socorro Island, Mexico. Pacific Conservation Biology 17: 11–21.